Among the methods developed up to the present time, stimulated emission depletion (STED) microscopy allows the imaging of the structures of a sample, which are marked with fluorescent probes, with a spatial resolution below the diffraction limit.
In a STED microscope, the excitation beam is spatially overlapped on a second de-excitation beam (also called a STED beam) which can de-excite, by stimulated emission, the fluorescent markers previously excited by the excitation beam. Since the STED beam has at least one zero intensity point (a diffused configuration uses a STED beam with a doughnut section having a zero intensity point at the centre), only the fluorescent markers in the proximity of the zero intensity points can actually emit fluorescence (that is to say, emit spontaneously) when they return to the ground state.
If the intensity of the de-excitation beam is increased, the probability of de-exciting the fluorescent species by stimulated emission increases, and the volume from which the fluorescent species emit decreases, reaching dimensions below the diffraction limit. When the two co-aligned beams are moved and the spontaneously emitted fluorescent light is recorded, the structures of interest marked with the fluorescent species are detected with a spatial resolution above the diffraction limit.
It is important to note that the probability of de-exciting a fluorescent species by stimulated emission depends not only on the intensity of the STED beam but also on the cross section of the stimulated emission process of the fluorescent probe at the wavelength of the STED beam: as the stimulated emission cross section increases, the probability of de-exciting a fluorescent species also increases. Therefore an increase in the stimulated emission cross section is immediately manifested in a reduction of the intensity of the STED beam required to achieve a given spatial resolution, thus reducing potential photodamage effects on the sample.
In STED microscopy, the wavelength of the STED beam is normally located near the red end of the emission peak of the fluorescent species. Shifting the wavelength of the STED beam nearer to the emission peak of the fluorescent species favourably increases the stimulated emission cross section. However, owing to the non-negligible overlap between the emission and excitation spectra, this shift also increases the probability of exciting, directly with the STED beam, the fluorescent species that have remained in the ground state, thereby causing undesirable background fluorescence. Although the fluorescent volume scanned by the excitation beam decreases, potentially improving the spatial resolution as a result, the fluorescent background generated by the STED beam increases the total effective fluorescent volume, thus seriously compromising the image contrast.
Fortunately, this undesired fluorescence background does not distort the spatial frequency content of the image, and in particular it does not alter the effective resolution of the image. In fact, the raw image consists of a super-resolved standard image (below the diffraction limit) generated by the excitation beam, plus a background image generated by the STED beam. Therefore, given a sufficient signal to noise ratio and reliable separate recording of the background image, the super-resolved background image can be retrieved simply by subtracting the recorded background image from the raw image.
It should be noted that, for a given STED wavelength, the possibility of subtracting the undesired background signal (generated by the STED beam) increases the choice of possible fluorescent species that can be used for imaging below the diffraction limit. As mentioned above, given a certain wavelength of the STED beam, it is only the fluorescent species with negligible excitation at the wavelength of the STED beam, and therefore a negligible background, that can conventionally be used successfully for STED imaging. Therefore the provision of a method for removing this background enables the spectral constraints to be relaxed for the fluorescent species, making this method highly important for multicolour imaging (a plurality of species with different spectral properties, each of which marks a different structure within the sample).
In view of the above, the present invention relates, in particular, to a method of optical microscopy by scanning a sample containing an excitable species, said method comprising, for each predetermined scanning segment:                directing a first light beam having a first main wavelength λ1, and a second light beam having a second main wavelength λ2, on to respective, partially overlapped areas of said sample, wherein said first light beam is provided for exciting, in single- or multi-photon excitation mode, members of the excitable species to an excited state, and said second light beam is provided for reducing the number of excited members in said excited state; and        detecting an optical signal coming from the sample, said optical signal comprising a main component emitted by the members excited by said first light beam, and a spurious component emitted by other members of said excitable species undesirably excited by said second light beam, wherein said optical signal is detected during consecutive first and second time gates having respective durations Topen and Tclose and defining a period T=Topen+Tclose, said first time gate being provided for detecting the optical signal for a time interval during which the main component and the spurious component are both present, and said second time gate being provided for detecting the optical signal for a time interval during which the main component tends to or is zero while the spurious component remains present;said method further comprising:        processing the optical signal detected during said second time gate for extracting an estimate of said spurious component; and        subtracting said estimate of the spurious component from the optical signal detected during said first time gate.        
A method of this kind, described in Vicidomini G et al. (2012), “STED with wavelengths closer to the emission maximum”, Opt. Express 20(5):5225-5236, uses the natural interruption of each pulsed laser to define the aforesaid time gates (the “open” and “close” phases). In particular, the time interval between two excitation pulses is divided into two time gates of equal length. The STED beam must operate in continuous wave (CW) mode or with a frequency twice as great as the frequency of the excitation pulses. Furthermore, the frequency of the excitation beam must be accurately chosen on the basis of the average lifetime τ of the excited state of the fluorescent species; in particular, the pulse interval must be sufficiently long to provide effective “open” and “close” phases (most of the fluorescent probes excited in a given phase must be de-excited during the same phase), but a longer pulse interval reduces the useful working cycle of the fluorescent species and only increases the noise. In the context of this scenario, the frequency of the excitation pulses must therefore vary according to the average lifetime of the fluorescent species, making the aforesaid method complicated and inflexible.
Moreover, since the average lifetime τ of the excited state of the fluorescent species is a few nanoseconds in most cases, in the aforementioned known method the “open” and “close” cycle must be completed in a few tens of nanoseconds. However, many detectors have a dead time (that is to say, the time after the recording of a photon during which the detector cannot record another photon) of the order of tens of nanoseconds. Therefore, when a photon is captured during the “open” phase with any probability, a potential photon reaching the detector in the subsequent “close” phase is not recorded, and therefore the background to be subtracted is under-estimated. The total dead time of the detection system increases further if a time-correlated single photon counting (TCSPC) card is used. Furthermore, the method in question is incompatible with STED microscopy based on a continuous wave excitation beam.
Another known method, described in Ronzitti E et al., (2013) “Frequency dependent detection in a STED microscope using modulated excitation light”, Opt. Express 21(1):210-219, uses a pulse generator to modulate the excitation beam at a frequency f and a lock-in amplifier (synchronized with the pulse generator) to selectively detect the fluorescence signal which follows the same frequency f. Since only the fluorescence signal generated by the excitation beam follows the same frequency, the fluorescence signal generated by the STED beam is eliminated by the filtering. The signal obtained from the lock-in amplifier is subsequently collected from an I/O card to form the final image. The lock-in amplifier detects the difference between the signal during the “open” phase of the modulation and the background during the “close” phase of the modulation. Since the pulse generator and the I/O card which control the scanning are independent, the scanning process and the modulation must be synchronized manually, thus reducing the versatility of the method described above.
Furthermore, the lock-in amplifier assumes that there are only “open” and “close” phases having the same duration, making it impossible to maximize the collection of the desired signal in the case of a weak background. Finally, the lock-in amplifier supplies only the signal filtered from the background; in other words, the “open” and “close” signals cannot be accessed separately, and therefore image post-processing methods cannot be used.